Gene therapy in the nervous system with superoxide dismutase

Neuronal death following necrotic insults involves the generation of reactive oxygen species (ROS). We investigated the effects of antioxidant gene therapy on ROS accumulation after exposure to either sodium cyanide, kainic acid or oxygen glucose deprivation (OGD). Specifically, we generated herpes simplex virus-1 amplicon vector expressing the gene for the antioxidant enzyme CuZnSOD. Overexpression of this gene in primary hippocampal cultures resulted in increased enzymatic activity of the corresponding protein. CuZnSOD significantly protected hippocampal neurons against sodium cyanide insult and the subsequent lipid peroxidation. However, it did not protect against OGD- or kainic-acid-induced toxicity. Moreover, CuZnSOD significantly worsened the toxicity, hydrogen peroxide accumulation and lipid peroxidation induced by kainic acid. As a possible explanation for this surprising worsening, CuZnSOD overexpression increased glutathione peroxidase activity in the presence of sodium cyanide but had no effect on catalase or glutathione peroxidase activity in the presence of kainic acid. Thus, cells were unlikely to be able to detoxify the excess hydrogen peroxide produced as a result of the CuZnSOD overexpression. These studies can be viewed as a cautionary note concerning gene therapy intervention against necrotic insults.     

CuZnSOD and MnSOD immunoreactivity in brain stem motor neurons from amyotropic lateral sclerosis patients

Motor neurons from the brain stems of amyotrophic lateral sclerosis (ALS) and control patients were examined with immunoantibodies to CuZn-superoxide dismutase (CuZnSOD) and Mn-superoxide dismutase (MnSOD). We found that there was a marked staining for CuZnSOD in all the motor nuclei, the hypoglossus, ambiguus, facialis and trigeminus from the ALS patients, but not in the controls. The same neurons from the ALS patients also stained very intensely for MnSOD, whereas the neurons from the control patients stained weakly or not at all. Loss of neurons was also a very consistent finding and was noted in all the motor nuclei from the ALS patients. There was a proliferation of glial cells which stained strongly both for CuZnSOD and for MnSOD accompanying the loss of the neurons. These results indicated that there was an apparent increase of superoxide dismutase immunoreactivity in motor neurons of ALS patients. We conclude that CuZnSOD and MnSOD immunoreactivity is increased in motor neurons and glia in the brain stems of patients with ALS, specific for the terminal phase of this disease. Received: 22 April 1997 / Revised, Accpted: 30 June 1997     

ApoE基因缺陷小鼠CuZnSOD基因CpG岛甲基化的研究

目的检测apoE基因缺陷小鼠肝脏CuZnSOD基因CpG岛甲基化状态,探讨其与动脉粥样硬化发生发展的关系以及在动脉粥样硬化早期基因诊断中的意义。方法ApoE基因缺陷小鼠是用来建立动脉粥样硬化的成熟模型,本实验将18例7周龄apoE基因缺陷雄性小鼠及作为对照的18例7周龄正常的C57BL/6J雄性小鼠分别随机分成三组,在饲养至0周、7周、14周时取其肝脏,运用甲基化特异性PCR（methylation-specific PCR,MSP）法检测肝脏CuZnSOD基因CpG岛甲基化情况。结果不同时间段apoE基因缺陷小鼠与正常的C57BL/6J小鼠CuZnSOD基因CpG岛甲基化差异无统计学意义。结论apoE基因缺陷小鼠肝脏没有发生CuZnSOD基因启动子区CpG岛的异常甲基化,可能CuZnSOD基因启动子区CpG岛的异常甲基化没有参与动脉粥样硬化的发生发展。     

Effects of variation in superoxide dismutases (SOD) on oxidative stress and apoptosis in lens epithelium

Among the critical antioxidant enzymes that protect the cells against oxidative stress are superoxide dismutases: CuZnSOD (Sod1) and MnSOD (Sod2). The latter is also implicated in apoptosis. To determine the importance of these enzymes in protection against reactive oxygen species (ROS) in the lens, we analysed DNA strand breaks in lens epithelium from transgenic and knockout (Sod1) mice following exposure to H2O2 in organ culture. Since Sod2 knockouts do not survive, comparison was made of lenses of partially-deficient (heterozygote) for Sod2 and the wild-type controls which have twice the enzyme level. Antioxidant potential of Sod2 was also studied in human lens epithelial cells (SRA01/04) in which the enzyme was up- and down-regulated by transfection with plasmids containing sense and antisense human cDNA for MnSOD. DNA strand breaks in the epithelium of Sod1 knockouts and Sod2 heterozygotes were much greater than in the corresponding wild-type or in transgenic mice over-expressing the enzymes when the lenses were exposed to H2O2. The functional role of Sod2 in apoptosis was examined in cultured human lens epithelial cells. Cells with higher enzyme levels were more resistant to the cytotoxic effects of H2O2, O2- and UV-B radiation. Furthermore, Sod2-deficient cells showed dramatic mitochondrial damage, cytochrome C leakage, caspase 3 activation and increased apoptotic cell death when they were challenged with O2-. Thus, mitochondrial enzyme (Sod2) deficiency plays an important role in the initiation of apoptosis in the lens epithelium.     

Effect of indometacin pretreatment on protamine sulfate-mediated relaxation of the isolated rat uterus: the role of the antioxidative defense system

Previous results in this laboratory indicate that protamine sulfate (PS) evokes dose-dependent relaxation of both spontaneous and calcium ion-induced uterus activity mediated predominantly by potassium channels and, to a small extent, via β-adrenergic receptors or nitric oxide (NO)-dependent pathways. Indometacin is a nonselective inhibitor of cyclooxygenase (COX 1 and COX 2) that has the ability to delay premature labor by reducing uterine contractions through the inhibition of prostanglandin synthesis in the uterus. This study investigates the effects of indometacin (0.1 and 1 μg/ml) pretreatment on the PS-induced relaxation of isolated uterine smooth muscle. Indometacin pretreatment per se did not change the activity of the uteri. However, indometacin significantly increased PS-induced relaxation of spontaneous uterine contractions. Indometacin pretreatment significantly decreased the magnitude and slope of PS-induced relaxation of calcium ion-induced uterine contractions. Indometacin pretreatment increased CuZnSOD activity and slightly increased GR activity during spontaneous uterine contractions when compared to PS alone. In calcium ion-induced contractions, indometacin pretreatment increased CuZnSOD, GSH-Px and GR activities. These results suggest that, in addition to its COX inhibitory effects, indometacin influences the effects of PS. Therefore, it is possible that indometacin regulates diverse cell functions via its association with lipid membranes by altering micro-environments within the membranes. The above-mentioned processes appear to be partly mediated by redox processes involving ROS, lipid peroxides and antioxidant enzymes. The extent of the PS-mediated effect as different in spontaneous versus calcium ion-induced active uteri.     

Protective Role of Aspirin,Vitamin C,and Zinc and their Effects on Zinc Status in the DMH-Induced Colon Carcinoma Model

Chemoprotection refers to the use of specific natural or synthetic chemical agents to suppress or prevent theprogression to cancer. The purpose of this study is to assess the protective effect of aspirin, vitamin C or zinc ina dimethyl hydrazine (DMH) colon carcinoma model in rats and to investigate the effect of these supplementson changes associated with colonic zinc status. Rats were randomly divided into three groups, group 1 (aspirin),group 2 (vitamin C) and group 3 (zinc), each being subdivided into two groups and given subcutaneous injectionof DMH (30 mg/kg body wt) twice a week for 3 months and sacrificed at 4 months (A-precancer model) and6 months (B-cancer model). Groups 1, 2, 3 were simultaneously given aspirin, vitamin C, or zinc supplementrespectively from the beginning till the end of the study. It was observed that 87.5% of rats co-treated with aspirinor vitamin C showed normal colonic histology, along with a significant decrease in colonic tissue zinc at bothtime points. Rats co-treated with zinc showed 100% reduction in tumor incidence with no significant change incolonic tissue zinc. Plasma zinc, colonic CuZnSOD (copper-zinc superoxide dismutase) and alkaline phosphataseactivity showed no significant changes in all 3 cotreated groups. These results suggest that aspirin, vitamin Cor zinc given separately, exert a chemoprotective effect against chemically induced DMH colonic preneoplasticprogression and colonic carcinogenesis in rats. The inhibitory effects are associated with maintaining the colonictissue zinc levels and zinc enzymes at near normal without significant changes.     

糖络宁对糖尿病大鼠坐骨神经MnSOD,CuZnSOD,GPx抗氧化酶基因表达的影响

目的:初步探讨中药复方糖络宁改善氧化应激的分子机制。方法:8周龄雄性SD大鼠随机选取15只为正常对照组,余大鼠禁食12 h后腹腔注射链脲佐菌素(STZ)60 mg·kg-1建立糖尿病模型,72 h后大鼠禁食8 h断尾取血,测定血糖≥16.7 mmol·L-1视为糖尿病(DM)模型建立成功。成功后随机分为模型组(M组)、糖络宁10 g·kg-1·d-1组(TLN组)、α-硫辛酸0.02 g·kg-1·d-1组(LA组)各15只,连续给药8周后,采用实时定量荧光PCR(Real-time PCR)技术检测各组大鼠坐骨神经锰超氧化物歧化酶(MnSOD)、铜锌超氧化物歧化酶(CuZnSOD)、谷胱甘肽过氧化物酶(GPx)基因表达量,动态监测血糖、冷热刺激反应时间和热痛阈值。结果:STZ建立糖尿病模型后,坐骨神经组织的抗氧化酶MnSOD,CuZnSOD,GPx基因表达均增加;糖络宁连续给药8周后,冷热刺激反应时间明显缩短(P<0.05),痛阈值明显延长(P<0.05),MnSOD,CuZnOD,GPx基因表达量均明显升高(P<0.05)。结论:糖络宁能够改善STZ糖尿病大鼠痛觉过敏、感觉迟钝症状,并能通过提高坐骨神经MnSOD,CuZnSOD,GPx基因表达减轻氧化应激。     

Biological definition of multiple chemical sensitivity from redox state and cytokine profiling and not from polymorphisms of xenobiotic-metabolizing enzymes

Background

Multiple chemical sensitivity (MCS) is a poorly clinically and biologically defined environment-associated syndrome. Although dysfunctions of phase I/phase II metabolizing enzymes and redox imbalance have been hypothesized, corresponding genetic and metabolic parameters in MCS have not been systematically examined.

Objectives

We sought for genetic, immunological, and metabolic markers in MCS.

Methods

We genotyped patients with diagnosis of MCS, suspected MCS and Italian healthy controls for allelic variants of cytochrome P450 isoforms (CYP2C9, CYP2C19, CYP2D6, and CYP3A5), UDP-glucuronosyl transferase (UGT1A1), and glutathione S-transferases (GSTP1, GSTM1, and GSTT1). Erythrocyte membrane fatty acids, antioxidant (catalase, superoxide dismutase (SOD)) and glutathione metabolizing (GST, glutathione peroxidase (Gpx)) enzymes, whole blood chemiluminescence, total antioxidant capacity, levels of nitrites/nitrates, glutathione, HNE-protein adducts, and a wide spectrum of cytokines in the plasma were determined.

Results

Allele and genotype frequencies of CYPs, UGT, GSTM, GSTT, and GSTP were similar in the Italian MCS patients and in the control populations. The activities of erythrocyte catalase and GST were lower, whereas Gpx was higher than normal. Both reduced and oxidised glutathione were decreased, whereas nitrites/nitrates were increased in the MCS groups. The MCS fatty acid profile was shifted to saturated compartment and IFNgamma, IL-8, IL-10, MCP-1, PDGFbb, and VEGF were increased.

Conclusions

Altered redox and cytokine patterns suggest inhibition of expression/activity of metabolizing and antioxidant enzymes in MCS. Metabolic parameters indicating accelerated lipid oxidation, increased nitric oxide production and glutathione depletion in combination with increased plasma inflammatory cytokines should be considered in biological definition and diagnosis of MCS.